Heme utilization by the enterococci

Abstract Heme consists of a tetrapyrrole ring ligating an iron ion and has important roles in biological systems. While well-known as the oxygen-binding molecule within hemoglobin of mammals, heme is also cofactor for several enzymes and a major iron source for bacteria within the host. The enterococci are a diverse group of Gram-positive bacteria that exist primarily within the gastrointestinal tract of animals. However, some species within this genus can transform into formidable opportunistic pathogens, largely owing to their extraordinary adaptability to hostile environments. Although enterococci cannot synthesize heme nor depend on heme to grow, several species within the genus encode proteins that utilize heme as a cofactor, which appears to increase their fitness and ability to thrive in challenging environments. This includes more efficient energy generation via aerobic respiration and protection from reactive oxygen species. Here, we review the significance of heme to enterococci, primarily the major human pathogen Enterococcus faecalis, use bioinformatics to assess the prevalence of hemoproteins throughout the genus, and highlight recent studies that underscore the central role of the heme–E. faecalis relationship in host–pathogen dynamics and interspecies bacterial interactions.


Introduction
Heme is an important nutrient cofactor for certain aerobe and anaerobe bacteria as it serves important roles such as energy generation, sensing and defense against reactive oxygen species (ROS), sensing of nitric oxide, as a trigger for enhanced biofilm formation, and can often be used as an iron source during host infection (Winstedt et al. 2000, Fr ankenber g et al. 2002, Pedersen et al. 2012, Choby and Skaar 2016, Layer 2021, Lee-Lopez and Yukl 2022 ).In mammals, heme is primarily found as the cofactor to hemoglobin within red blood cells, but it is also found in abundance as a cofactor in myoglobin within muscle cells and many other proteins (Ponka 1999 ).Additionally, levels of heme within the gut of mammals and other animals v ary widel y depending on the diet (Khalili et al. 2017 ).The v aried bioav ailability and distribution of heme within the host makes mechanisms for heme acquisition or synthesis, utilization, and e v en detoxification important fitness factors for some species of commensal, opportunistic, or pathogenic bacteria (Gruss et al. 2012 , Baureder andHederstedt 2013 ).Se v er al r e vie ws hav e highlighted the r ole of heme in bacterial physiology to include the importance of hemophores to the evolution of the oral and gut microbiota (Olczak et al. 2024 ), the heme synthesis pathways (Yang et al. 2024 ), and heme acquisition and tolerance mechanisms in Gram-positive bacteria (Wang et al. 2023 ).
The enter ococci ar e gut dwelling Gr am-positiv e diplococci that arose 425-500 million years ago and have been coevolving in the guts of animals for millions of years (Lebreton et al. 2014(Lebreton et al. , 2017 ) ). Curr entl y, the Enterococcus genus comprises a little over 60 species and r epr esentativ es ar e found in the guts of virtually all animals including insects , aquatic animals , reptiles , amphibians , birds , and mammals (Lebreton et al. 2014(Lebreton et al. , 2017 ) ).In addition to being gut commensals, Enterococcus faecalis and Enterococcus faecium , the two major human colonizers, are also opportunistic pathogens that surviv e and toler ate m ultiple str essors commonl y found within the host and hospital environments (Lebreton et al. 2014, 2017, Schwartzman et al. 2023 ).These stressors often include low nutrient availability in the environment, host-imposed starvation, fierce competition with other microbes for available nutrients, fluctuations in environmental pH and osmolarity, disinfectants such as chlorhexidine, and production of ROS or antimicrobial peptides by the host or competing microbes (Gaca and Lemos 2019, García-Solache and Rice 2019, Gaston et al. 2020 ).While the enterococci do not require heme for growth nor can they synthesize their own heme, they do possess heme-dependent enzymes that can aide the cell in overcoming many of these stressors including more efficient energy generation, protection against ROS, and utilization of heme as an iron source (Fig. 1 ) (Winstedt et al. 2000, Fr ankenber g et al. 2002, Brunson et al. 2023 ).
Most of what is known concerning heme utilization in enterococci comes from studies performed with E. faecalis and include identification of heme-dependent catalase and cytoc hr omes, heme-binding tr ansmembr ane pr oteins, and c har acterization of a heme-sensing regulator and heme export system (Winstedt et al. 2000, Fr ankenber g et al. 2002, Baureder et al. 2012 ).Additionally, a heme-binding protein annotated as either a copr opor phyrinogen III oxidase or a heme c ha per one has been identified but not c har acterized (Wilkinson et al. 2023 ).This r e vie w will primarily focus on the significance of hemoproteins to the physiology and virulence of E. faecalis.Considering that very little is known about heme utilization by other enterococci, we also used bioinformatics to assess the pr esence/pr e v alence of eac h E. f aecalis hemopr otein to potential homologues in other enterococci.

Figure 1.
Established and predicted mechanisms of heme acquisition, utilization, and export in enterococci.Heme is an essential cofactor of cytoc hr ome bd and catalase and can be an important source of iron within the host.Toxicity is managed by a heme-sensing regulator FhtR and an export system HrtAB, but import is likely mediated by CydCD and other unidentified import systems(s) or through passive diffusion across the membrane.Heme has also been established as an important molecule in microbe-microbe and host-microbe interactions in which hemoproteins of host and bacterial origin are degraded and the freed heme is used by E. faecalis to enhance biofilm formation.Created using BioRender.

Energy gener a tion by cytoc hr ome bd
Enter ococci primaril y deriv e ener gy fr om fermentation of sugars with only a few species having an additional capacity to conserv e ener gy via aer obic r espir ation (Ramsey et al. 2014 ).One suc h species, E. f aecalis, can activ el y r espir e, but onl y in the presence of exogenous heme that is the cofactor of the r espir atory enzyme complex cytoc hr ome bd .The E. f aecalis cytoc hr ome bd consists of two polypeptides, CydA and CydB.The CydA protein contains the three distinct heme-binding domains, cytochrome b 558 , which is the site of quinol oxidation, as well as b 595 and d (Borisov et al. 2021 ) .The oxidase activity of the enzyme generates a transmembr ane gr adient that ultimatel y driv es ATP ase activity.The second polypeptide is encoded by cydB , and while it does not contain heme, it is belie v ed to be par alogous to cydA having arisen from a duplication e v ent (Cook and Poole 2016 ).Together, CydA and CydB form a heterodimeric complex that is stabilized by interactions of alpha helices α3, α4, and α9 (Borisov et al. 2021 ).Importantly, supplementation with heme to the growth media has been shown to enhance both planktonic gr owth (P ainter et al. 2017 ) and biofilm formation in E. faecalis (Ch'ng et al. 2022 ).
The Bacteria and Viral Bioinformatics Resource Center (BV-BRC) combines se v er al tools and databases for bacterial bioinformatic analyses (Olson et al. 2023 ).As of March of 2024, this database contained whole genome sequences of 66 enterococci isolated from environmental, animal, or human sources.T hroughout this article , we utilized this database to search for homologues of E. faecalis hemoproteins in other members of the genus to gain a broader insight into the significance of heme to other enterococci.For these analyses, we only included enterococcal species in which there were at least five genomes with good quality sequencing, which gave us 25 species of enterococci to search for hemoproteins .T he E. faecalis OG1RF genome was used as the basis for most of our comparisons because it is the most used strain for E. faecalis genetic manipulations and gener all y accepted as a good r epr esentativ e of the species (Bourgogne et al. 2008, Dale et al. 2018 ).In addition, we included the closel y r elated (and former enterococci) Vagococcus , Melissococcus , and Tetragenococcus species (Lebreton et al. 2014, 2017, Schwartzman et al. 2023 ).A positive hit was considered if the protein identified shar ed gr eater than 50% amino acid identity and greater than 90% query and subject cov er a ge.First, we pr obed the pr e v alence of CydA (OG1RF_RS08540) and CydB (OG1RF_RS08535) across strains of E. faecalis and other species of enterococci.We found that nearly all strains of E. faecalis (98%-99%) encode CydA and CydB while both genes are completely absent in E. faecium strains.Overall, the distribution of cytoc hr ome bd in the genus is une v en with se veral species (e.g.E. avium , E. casseliflavus , E. cecorum , and E. gallinarum ) encoding CydA and CydB homologues while other species including E. durans , E. hirae , and E. lactis do not.Additionally, we found a high pr e v alence of CydA and CydB in Tetr a genococci and Va gococci, while both pr oteins can be found r ar el y in Melissococci (Fig. 2 ).
Figure 2. Phylogenetic relationships of the enterococci and prevalence of proteins involved in heme utilization.The BV-BRC database was searched for enterococcal species with at least five sequenced genomes of good quality.Re presentati ve genomes were chosen for phylogenetic tree construction using the BV-BRC bacterial genome tree tool.The total number of genomes used to search for potential hemoprotein homologues are shown.A genome was considered positive for a particular hemoprotein if the query and subject cov er a ge wer e > 90% and the amino acid identity was > 50%.The percentage of species that was found to be positive for each protein is indicated numerically and by heat-map, where darker colors indicate proteins ar e mor e pr e v alent within isolates of the species.* Indicates the pr esence of a potential homologue with 40%-50% amino acid identity.The phylogenetic tree was constructed using the BV-BRC bacterial genome tree tool.A re presentati ve genome from each species was chosen and 1000 genes were used in the Codon Tree method in which both amino acid and DNA sequences from single copy genes were analyzed using RAxML.The most common host(s) each species was isolated from is shown.Created using BioRender.

T he tr ansmembr ane pr oteins CydC and CydD are associated with cytochrome bd assembly and heme uptake
The cydA and cydB genes are part of an operon, cydABCD , in which c ydC and c ydD encode integral membrane proteins each with an ATPase domain.In Escherichia coli , the CydCD complex is required to export thiol-containing compounds, uses heme as a cofactor to support its ATPase activity, and is r equir ed for cytoc hr ome bd assembly (Pittman et al. 2002, Yamashita et al. 2014, Holyoake et al. 2015, 2016, Shepherd 2015, Poole et al. 2019 ).Recently, it has also been demonstrated that the CydCD complex of E. coli captures heme from the cytoplasmic membrane and uses a trapand-flip mechanism to transport heme to the periplasmic space (Wu et al. 2023 ).In E. faecalis , CydCD w as also sho wn to be requir ed for cytoc hr ome bd assembl y (Baur eder and Hederstedt 2012 ) and was linked to heme transport as intracellular heme w as belo w the detection limits in whole cell l ysates of tr ansposon mutants disrupted in either cydC or cydD (Ch'ng et al. 2022 ).Accordingly, the disruption of cydC or cydD also led to a defect in biofilm biomass when grown in the presence of heme (Ch'ng et al. 2022 ).Taken together, these data suggest that CydCD is a major driver of heme uptake in E. faecalis and essential for activation of the c ytochromes.Ho w ever, a few points remain unclear.First, in contrast to E. coli CydCD, which was shown to transport heme from the cytoplasmic membrane to the periplasmic space, E. f aecalis CydCD a ppears to play a r ole in heme import.While it lac ks v alidation, it is tempting to speculate that extracellular heme becomes associated with E. f aecalis membr anes and then is transported to the c ytoplasm b y a similar tr a p-and-flip mec hanism.Second, str ains lac king CydCD r etain catalase activity, an enzyme whose function is reliant upon the presence of intracellular heme (Baureder and Hederstedt 2012 ).Retention of catalase activity indicates the presence of alternate heme import mec hanisms that hav e yet to be identified.Notabl y, enter ococcal genomes do not encode homologues of well-c har acterized heme scavenging and import systems found in related bacteria such as Streptococcus pyogenes or Staphylococcus aureus , making the identifi-cation of heme importers in the enterococci a difficult endeavor (Cassat and Skaar 2012, Ouattara et al. 2013, Pishc han y et al. 2014, Zhang et al. 2017, Chatterjee et al. 2020, Wang et al. 2023 ).Considering the r ele v ance of heme to bacterial pathophysiology, the identification of the heme importers of E. faecalis should be a priority in the field.Like cytoc hr ome bd , both CydC (OG1RF_RS08525) and CydD (OG1RF_RS08530) were found in almost all E. faecalis strains, but not in E. faecium, and most species that possess CydA and CydB also had a pr e v alence for CydC and CydD (Fig. 2 ).

Protection against o xidativ e stress by catalase
Except for E. f aecalis , the enter ococci ar e gener all y classified as catalase negativ e (Baur eder and Hederstedt 2013 ).Pr e vious studies have demonstrated that heme is an essential cofactor for catalase in E. faecalis and that catalase protects against exogenous H 2 O 2 but not when H 2 O 2 is produced endogenously as a byproduct of gl ycer ol metabolism (Clarke and Knowles 1980, Pugh and Kno wles 1983, F r ankenber g et al. 2002, Baur eder et al. 2012 ).Regulation of the katA gene is under control of the HypR and Spx tr anscriptional activ ators, particularl y in H 2 O 2 str essed cells, but is independent of heme availability (Verneuil et al. 2005, Kajfasz et al. 2012, Baureder et al. 2014 ).It has also been shown that the catalase polypeptide (KatA) accumulates in E. faecalis during exponential phase independent of heme, but heme-supplemented cultures contain almost double the amount of catalase as nonsupplemented cultures.During stationary growth phase, the KatA pol ypeptide is degr aded in the absence of heme while it remains stable in heme-rich cultures (Frankenberg et al. 2002, Baureder et al. 2014 ).Taken together, these observations indicate that heme is not only essential for activity but also stabilizes the catalase protein.
The E. faecalis catalase is a homotetrameric protein with each subunit binding one molecule of heme and a proline residue (P28) has been indicated as being essential to its activity (Hakansson et al. 2004, Baureder and Hederstedt 2012, Baureder et al. 2014 ).While the structure of catalase in E. faecalis has been solv ed, ther e is still a limited understanding regarding heme trafficking or how catalase assembly occurs in vivo (Brugna et al. 2010, Baureder et al. 2014 ).A tr ansposon m utant libr ary was used to screen for potential genes involved in catalase biogenesis including heme chaper one(s) and tr ansporter(s) (Baur eder and Hederstedt 2012 ).Fiv e loci appear to indirectly impact catalase activity in E. faecalis , including rnjA and srmB , both of whic h ar e involv ed in RNA turnov er (Condon 2010, Roux et al. 2011 ), npr which encodes for an NADH peroxidase important for endogenous H 2 O 2 detoxification (La Carbona et al. 2007, Wasselin et al. 2022 ), the stress and virulence regulator etaR (Teng et al. 2002 ), and the oligopeptide transporter oppBCDF , which has been proposed to moonlight as a low affinity heme transporter (Letoffe et al. 2006 , Baureder andHederstedt 2012 ).Future investigations into the OppBCDF transporter capacity to import heme, alone or in conjunction with CydCD, and their roles in virulence should be investigated further.Other than katA , no single gene was essential for catalase function as all m utants r etained some le v el of activity, suggesting that heme importers and c ha per ones wer e either missed in the scr een or are encoded by multiple functionally redundant genes (Baureder et al. 2014 ).
While most strains of E. faecalis encode a heme-dependent catalase (catalase h ), a manganese-dependent catalase has been identified and partially characterized in E. faecium (catalase Mn ).Ho w e v er, the catalase Mn does not appear to protect E. faecium from either endogenous or exogenous H 2 O 2 stress (Wasselin et al. 2022 ).We used the BV-BRC database to search for homologues of E. faecalis OG1RF catalase h (OG1RF_RS06790) and E. faecium DO catalase Mn (HMPREF0351_10525) in other enterococci.Of the 25 species, onl y thr ee species other than E. f aecalis wer e found to encode a catalase h , albeit with varying degrees of prevalence.Specifically, catalase h was found in 26% of E. casseliflavus , 3% of E. gallinarum , and 83% of E. sacc harol yticus str ains, and in no E. f aecium str ains, confirming pr e vious r esults (Baur eder and Hederstedt 2013 ) (Fig. 2 ).Inter estingl y, se v er al of the species of enterococci that did not encode catalase h had a high degree of pr e v alence for catalase Mn , including E. faecium (74%), E. avium (92%), E. durans (98%), and E. gallinarum (92%) (Fig. 2 ).A small percentage (7%) of E. faecalis strains also encoded a catalase Mn and 97% of E. casseliflavus encoded catalase Mn , indicating that some strains of E. faecalis and E. casseliflavus possess both types of catalases.In Vagococci , 1% of the sequenced strains encoded catalase h and no strains were found to encode catalase Mn , in Tetragenococci , 8% encode catalase h and 33% encoded a catalase Mn homologue sharing 40%-50% amino acid identity with E. faecium catalase Mn , and no Melissococcus strains encode either type of catalase.Future studies should confirm catalase activity and determine the mechanism of detoxification of catalase Mn , inv estigate the e volutionary significance of catalase h and catalase Mn in different enterococcal species, and determine possible differences in the contribution to bacterial fitness of catalase h and catalase Mn in different environments the enterococci inhabit.

Heme toxicity is managed by an export system and heme sensing regulator
Despite heme being a nutrient cofactor, intracellular heme le v els m ust be tightl y contr olled, as heme can be toxic to cells at high concentr ations.In E. f aecalis OG1RF, heme toxicity is managed by an ABC-type heme efflux transporter HrtAB (OG1RF_RS02770-RS02775) and heme-sensing TetR family regula-tor FhtR (OG1RF_RS02765) (Saillant et al. 2021 ).FhtR ( E. f aecalis heme tr ansport r egulator) exerts negativ e contr ol ov er hrtAB in a heme-dependent manner; when intracellular heme levels rise, heme-bound FhtR is r eleased fr om the hrtAB promoter region and transcription is activated (Saillant et al. 2021 ).The heme exporter is comprised of two subunits r eferr ed to as HrtA and HrtB for heme regulated transport.HrtA is the ATPase and HrtB is an FtsX-like permease.Expression of hrtAB is higher in the presence of heme, hemoglobin, and blood as well as within the gastrointestinal tract of mice suggesting that heme efflux might be important to E. faecalis during systemic infections and gut colonization.HrtAB is a member of the MacB family of efflux pumps in which instead of exporting heme dir ectl y fr om the cytoplasm, HrtB binds to heme tr a pped in the membrane and HrtA uses ATP to change confirmation of the heme bound complex to release heme into the extracellular environment (Nakamura et al. 2022 ).Due to the lipophilic nature of heme, it is possible heme may be able to passiv el y diffuse acr oss the cell membr ane (Donegan et al. 2019 ).Similar to E. faecalis , Lactococcus lactis expresses an HrtAB heme exporter and no high affinity heme import system has been described (Joubert et al. 2014 ).Se v er al studies have thus suggested that in some Gr am-positiv e bacteria, ther e is not a need for an active heme uptake system, but that heme can become membrane associated, diffuse across the membrane, or be r emov ed via an HrtAB type exporter (Joubert et al. 2014, Saillant et al. 2021, Nakam ur a et al. 2022 ).In conjunction with the r ecent discovery that CydCD binds and transports heme from the membrane, it is worth considering that in E. faecalis , heme is not scavenged from the environment via high affinity heme transporters, but rather heme becomes associated with the membrane, diffuses acr oss, is tr ansported via CydCD, or is exported by HrtAB (Joubert et al. 2014, Saillant et al. 2021, Nakam ur a et al. 2022, Wu et al. 2023 ).
Using BV-BRC and the same criteria as before, we found no other enterococci encoding FhtR.In a pr e vious study, the E. faecalis FhtR was found to be more closely related to orthologues found in Vagococci (we found four species with > 59% amino acid identity) and Carnobacterium sp than to potential orthologues in E. faecium (we found strains with < 40% amino acid identity), E. hirae (we found strains with < 40% amino acid identity), or E. avium (we found strains with < 40% amino acid identity) (Saillant et al. 2021 ).In contrast to FhtR, we identified orthologues of HrtA ( > 50% amino acid identity) and an associated permease with lo w er similarity to HrtB (40%-50% amino acid identity) throughout several species of enterococci (Fig. 2 ).Using the E. faecium DO reference genome, we found that the most similar ATPase (58.6% amino acid identity) and permease (48% amino acid identity) components to HrtAB were indeed genetically linked to each other and to a TetR-type regulator (HMPREF0351_11396-11398) sharing 14.7% amino acid identity with the E. faecalis FhtR.It is important to note that both E. faecalis and E. faecium can tolerate high levels of heme in upw ar ds of 50 μg/ml ( ∼74 μM heme) (Ok or oc henk ov et al. 2013 ).T hus , it is possible that E. faecium , and possibly other enterococci, possess interconnected heme efflux/heme-sensing systems orthologous or analogous to HrtAB/FhtR.

Heme may be an important source of iron for E. faecalis in systemic infections
Considering that heme is the most abundant form of iron in mammalian hosts, it is unsurprising that some of the most successful pathogenic bacteria employ multiple and diverse strategies to ob-tain heme from the environment to use as an iron source (Runyen-J aneck y 2013 , Sheldon and Heinrichs 2015, Choby and Skaar 2016, Hare 2017, Chatterjee et al. 2020, Jochim et al. 2020, Flannagan et al. 2022 ).Recentl y, our gr oup sho w ed that E. faecalis encodes at least fiv e ir on uptak e systems that, collecti v el y, ar e important for host colonization but that ir on starv ation in a strain lacking all five ir on tr ansporters can still be ov er come b y heme supplementation (Brunson et al. 2023 ).While the mechanisms of heme acquisition in E. f aecalis r emain elusiv e , as discussed abo ve , results from this in vestigation lea ve little doubt that heme is an important source of iron to E. faecalis during infection.
Before heme can be used as an iron source, the iron atom must first be liberated from the porphyrin ring, which in bacteria is typically accomplished by a family of enzymes known as bacterial heme oxygenases (Lyles and Eichenbaum 2018 ).Howe v er, E. f aecalis does not encode any of the c har acterized canonical or noncanonical heme oxygenases.On the other hand, all E. faecalis genomes encode a potential orthologue (OG1RF_RS05575) of a r ecentl y described family of enzymes identified in Gramnegative bacteria, known as anaerobilin synthase, that utilize radical S -adenosylmethionine methyltr ansfer ase to open the porphyrin ring and free the iron ion (LaMattina et al. 2016, Mathew et al. 2019, 2022, Brimberry et al. 2021 ).The best-c har acterized enzyme of the family, ChuW, was first described in E. coli and shares ∼44% amino acid identity with E. faecalis RS05575.While the E. faecalis ChuW orthologue is often annotated as a copr opor phyrinogen synthase (anaerobic heme synthesis) or as a heme chaperone, we suspect RS05575 functions as a heme-degrading enzyme under anaerobic conditions.Our lab is curr entl y inv estigating the potential functions of RS05575, including heme degradation and c ha per one activities.Mor eov er, we found that all species of Enterococcus , Vagococcus , Melissococcus , and Tetragenococcus encode an orthologue of RS05575 with varying degrees of prevalence (40%-100%), which suggests that heme degradation or other functions of this enzyme are advantageous to all enterococci and closely r elated gener a that r eside in the guts of animals.
In addition to this putative anaerobilin synthase, some enterococcal isolates encode hemH , a ferrochelatase whose exact function in the enterococci is unknown.In some bacteria, ferr oc helatases ar e involv ed in heme biosynthesis (Choby and Skaar 2016 ) while in anaerobes such as P orph yromonas gingivalis , ferr oc helatase (encoded by ihtB ) was shown to degrade extracellular heme to liberate the iron atom (Dashper et al. 2000 ).We speculate that like the P. gingivalis IhtB, the enterococcal HemH homologue works in the r e v erse r eaction and does not synthesize heme but r ather degr ades it due to the absence of other genes in the heme biosynthetic pathway in their genomes.In a study using comparative genomics to identify bacterial fitness factors in gut, urinary tract, and blood isolates, hemH was found in isolates from all three sites, but enriched in urinary tract isolates compared to gut and blood isolates (Sharon et al. 2023 ).The mechanistic function and role of E. faecalis HemH in virulence in different host niches should be investigated to gain a better understanding of when or if this auxiliary protein enhances virulence or interspecies competition.We used HemH (RS09495) fr om E. f aecalis V583 to searc h for potential homologues in other enterococci and found that HemH is present in 62% of E. f aecalis str ains, 96% of E. cecorum , 100% of E. dongliensis , and E. hulanensis , and 52% of the Vagococci (Fig. 2 ).Yet, se v er al other enter ococci, including E. f aecium , do not encode HemH.This une v en distribution among the enter ococci begs the question of what role does HemH play in enterococcal niche adaptation and why is it absent in some species but highly prevalent in others?

Other hemoproteins of enterococci
Despite the significance of heme as a cofactor and iron source to bacteria, the heme-binding proteome is not well-defined.This includes canonical heme proteins that bind heme with a high affinity and r equir e heme for activity or that tr ansport heme, r eferred to as heme proteins, and those that transiently bind heme or bind heme with low affinity, but heme is not essential for activity, r eferr ed to as heme-binding proteins .T he development of click chemistry in which proteins are specifically labeled using biorthogonal chemical reactions combined with the po w er of lar ge-scale pr oteomics has r ecentl y helped expand the number of known heme proteins and heme-binding proteins in bacteria (Parker and Pratt 2020 , Wilkinson et al. 2023 ).This method was used to label heme with different probes to identify heme-binding pr oteins in E. f aecalis V583 with fiv e pr e viousl y c har acterized pr oteins, namely HrtA, HrtB, CydA, CydC, and CydD, confirmed to have the capacity to bind heme (Wilkinson et al. 2023 ).Additionall y, se v er al ne w potential heme pr oteins and heme-binding pr oteins wer e identified, including the putativ e anaer obilin synthase OG1RF_RS05575 mentioned abo ve , the per oxir edoxin AhpC that protects cells from organic peroxide and H 2 O 2 stressors, and the type I gl ycer aldeh yde-3-phosphate deh ydrogenase (GAPDH).While heme was not anticipated to interact with AhpC (La Carbona et al. 2007 )-the solved crystal structure of E. faecalis AhpC provides no indication of a heme-binding motif (Pan et al. 2018 )-GAPDH has been shown to bind heme in mammalian cell lines (Chakr av arti et al. 2010 ) and purified GAPDH from the lactic acid bacteria Streptococcus gordonii and Streptococcus suis was shown to bind heme in vitro (Hannibal et al. 2012, Slezak et al. 2020 ).Furthermor e, four pr oteins with no pr e vious association with heme wer e also pulled down using heme-based probes including the copper c ha per one CopZ, an N -acetylm ur amoyl-l -alanine amidase, a dala-d -ala carbo xype ptidase, and a tr anscriptional r egulator fr om the UvrC family.Potential heme-binding motifs within each of these proteins was determined using the HeMoQuest web server but these predictions still need to be experimentally validated (P aul Geor ge et al. 2020, Wilkinson et al. 2023 ).In the future, this po w erful system can be refined to expand the list of heme-binding pr oteins in enter ococci by assaying under differ ent envir onmental conditions .T his should include under aer obic v ersus anaer obic conditions, extreme iron starvation, planktonic versus biofilm lifestyles, and in assessing cell wall associated and secreted proteins.

Enterococcus faecalis polymicrobial and host-pa thogen inter actions are influenced by heme
Because enterococci are natural residents of the complex gut micr obiota and ar e fr equentl y isolated fr om extr a intestinal sites in pol ymicr obial infections, it is important to also consider the role heme and hemoproteins play in cross-bacterial and hostpathogen interactions.On this note, two recent studies have shed new light into the importance of heme as a metabolite in E. faecalis interspecies and host interactions.In the first study, the Kline lab sho w ed that E. faecalis produces significantly more biofilm when grown in mixed cultures with S. aureus (Ch'ng et al. 2022 ).This phenomenon w as sho wn to be associated with the presence of both S. aureus and E. faecalis factors.Specifically, S. aureus hemopr oteins wer e degr aded by secr eted E. f aecalis gelatinase, whic h resulted in heme-mediated activation of aerobic respiration by cytoc hr ome bd.The authors concluded that aerobic respiration enhanced biofilm formation by increasing the ATP supply necessary for the synthesis of ener gy costl y biofilm matrix and adhesion pr oteins suc h as EpaOX and EbpABC.Furthermor e, secr eted gelatinase was shown to break down hemoglobin, providing E. faecalis with a heme source of host origin (Ch'ng et al. 2022 ).
In the other study, the Zackular lab sho w ed that coinfection of the mouse gut with Clostridioides difficile enables E. faecalis to access both heme and oxygen in the otherwise anoxic and low heme gut envir onment, r esulting in r obust interspecies biofilms (Khalili et al. 2017, Smith et al. 2024 ).This interspecies cooperation is accomplished by C. difficile production of toxins that damage the gut epithelium leading to an influx of hemoglobin/heme and diffusion of oxygen into the gut.As a r esult, E. f aecalis utilizes heme and oxygen for aerobic respiration, which leads to increased biofilm formation and ov er all fitness gain, a phenotype that is reliant upon E. faecalis CydAB (Smith et al. 2024 ).In turn, E. faecalis assists C. difficile by metabolizing intestinal arginine (via the arginine deiminase system) and secreting ornithine, which promotes C. difficile colonization and increased toxin production (Smith et al. 2022 ).This synergistic relationship results in increased severity of infection and poor outcomes in patients with C. difficile colitis who are also heavily colonized with E. faecalis , particularly VRE strains (Smith et al. 2022(Smith et al. , 2024 ).Yet, an E. f aecalis tr ansposon m utant lac king c ydA ( c ydA ::Tn) display ed no fitness defect in a mouse single species gut colonization model, indicating that under normal circumstances aer obic r espir ation is not a fitness factor for E. faecalis within the gut (Smith et al. 2024 ).
Notabl y, two separ ate studies hav e uncov er ed that E. faecalis engages in extracellular electron transfer (EET) as a method for ener gy gener ation, distinct fr om heme and cytoc hr ome activ ation (Keogh et al. 2018, Pankratova et al. 2018, Hederstedt et al. 2020 ).Mor eov er, it has been shown that heme negativ el y impacts ferric reductase activity of E. faecalis EET, a phenomenon that is relie v ed in a cytoc hr ome-deficient str ain (Hederstedt 2022 ).While E. faecalis EET has yet to be demonstrated in vivo , a recent study with Listeria monocytogenes showed that an EET-deficient mutant displayed a competitive fitness defect in the mouse gastrointestinal tract (Light et al. 2018 ).T hus , it is possible that E. faecalis can also utilize EET to gener ate ener gy within the gut, pr ovided heme and oxygen are in low enough levels to prevent cytochrome activation.
In contrast to the studies discussed abo ve , another group found that growing E. faecalis in the presence of heme led to sensitization to the o xidati v e burst of neutr ophils (P ainter et al. 2017 ), indicating that aerobic respiration may be detrimental to E. faecalis at sites with high neutrophil recruitment.Ho w ever, this study was performed with planktonic cultures and failed to consider if cells within a biofilm can be protected from these effects .T herefore , it is possible that at infection sites where E. faecalis persists in a pol ymicr obial biofilm lifestyle, such as the wound bed or on the surface of a urinary catheter, heme acquisition, and aerobic respiration may enhance the virulence of E. faecalis like what was observed in cocolonization with C. difficile in the gut.
Heme is both necessary and cytotoxic to the mammalian host due to its ability to promote and pr opa gate the formation of danger ous ROS, particularl y the ca pacity of heme to cause lipid peroxidation of eukaryotic membranes (Kumar and Bandy opadhy ay 2005 ).T hus , just as free iron and other metals ar e tightl y r egulated by the host at cellular and systemic le v els to avoid toxicity, so is heme.In mammals, free hemoglobin is quickly bound by ha ptoglobin, a pr otein that tr affics hemoglobin to hepatocytes and macr opha ges for degr adation (Andersen et al. 2017, Mozzi et al. 2018 ) while any free heme is sequestered by hemopexin and tr affic ked to the liver for catabolism (Morgan 1976, Tolosano et al. 2010 ).Expression of both haptoglobin and hemopexin are induced by IL-22 in response to inflammation during infection to restrict the availability of heme as a nutrient and iron source to bacterial pathogens (Datta 2017, Sakamoto et al. 2017, Mozzi et al. 2018 ).As of no w, ho w enterococci compete for access to heme during infection remains largely unknown for several reasons, many of which have been discussed abo ve .First, the major drivers of heme acquisition in enterococci are still poorly understood.Second, while the degradation of host and bacterial hemoproteins by E. faecalis gelatinase was shown to be important for heme acquisition, this phenomenon was not observed in the majority of strains e v aluated, including str ains positiv e for gelatinase.Mor eov er, at least two strains that lacked gelatinase activity were found to retain the ability to use hemoglobin to enhance biofilms (Ch'ng et al. 2022 ).Notably, we found that 84% of E. faecalis strains in the BV-BRC database encode gelE (OG1RF_07835) but no other species of enterococci as well as Vagococci, Melissococci, or Tetragenococci can produce gelatinase (Fig. 2 ).All together this is highly suggestive that while gelatinase might play a significant role in liber ating heme fr om hemopr oteins in E. f aecalis , it is clearl y not the sole mechanism.We propose there must be alternative ways that E. faecalis can access heme from hemoproteins.One possible mechanism is through the production of H 2 O 2 , which oxidizes hemoglobin to methemoglobin causing the release of heme from the protein, a process that has been demonstrated in Streptococcus pneumoniae (McDevitt et al. 2020 ).Furthermore, extended incubation of heme with H 2 O 2 -pr oducing S. pneumoniae r e v ealed that this pr ocess can e v entuall y lead to heme degr adation (Aliba yo v et al. 2022, Womack et al. 2024 ).Of note, E. faecalis generates low levels of H 2 O 2 that is enhanced when grown on alternative sugars such as glycerol and galactose and during severely dysregulated metabolism, likely due to superoxide dismutase conversion of hydr oxyl r adicals to H 2 O 2 (La Carbona et al. 2007, Colomer-Winter et al. 2017 ).T hus , it is conceiv able that E. f aecalis may also r el y on net production of H 2 O 2 to release and subsequently degrade heme fr om hemopr oteins.

Hemolysins of enterococci
The points discussed above indicate that E. faecalis can passively utilize free heme or liberate heme from hemoproteins, but little is r eall y understood r egarding additional a ppr oac hes that may be taken by E. faecalis or other enterococci to increase bioavailable heme, such as the production of hemolysins.Lysis of red blood cells by hemolysins increases a wealth of nutrients available to enterococci, including heme.In general, the Enterococcus genus is categorized as gamma hemol ytic, indicativ e of weak or no hemolysis, although enterococcal strains displaying either alpha or beta hemolysis have been isolated from clinical studies (Semedo et al. 2003, Creti et al. 2004, Gok et al. 2020, Hashem et al. 2021, Zarzecka et al. 2022 ).In fact, plasmids or c hr omosomal pathogenicity-associated islands have been shown to possess noncore genes, known as cytolysins, associated with hemolysis and/or killing of other Gr am-positiv e bacteria (Shankar et al. 2004 ).Cytolysin genes include two operons that are genetically linked but transcribed in opposite orientation.The regulatory genes cylR1 and cylR2 are transcribed in the leftw ar d direction and the structural genes c ylL(L) , c ylL(S) , c ylM , c ylB , c ylA , and cylI ar e tr anscribed in the rightw ar d direction.The c ylL(L) and cylL(S) genes encode immature peptides that form the active cytolytic toxin.The cylM product post-translationally modifies the immature peptides prior to secretion, and the cylB gene product is localized to the membrane and processes the polypeptide .T he cylA gene codes for another secreted protein that cleaves the peptides to form the mature toxin and cylI encodes an immunity protein that protects the parent cell from the toxin (Shankar et al. 2004, Van Tyne et al. 2013 ).Acr oss se v er al clinical studies, the association of hemolytic E. faecalis isolated at different infection sites has demonstrated a higher pr e v alence of cytol ysin positiv e E. f aecalis from nonsystemic infections such as wound or urinary tract infections than from systemic infections such as bacteremia or infectiv e endocarditis (Huyc ke and Gilmor e 1995, Arc himbaud et al. 2002, Creti et al. 2004 ).Conversely, one such study demonstrated an enrichment of hemolytic enterococci from the gut of hospitalized patients compared to enterococci isolated from healthy controls in the community (Creti et al. 2004 ).Taken together this may indicate that while the hospital environment selects for hemolytic enterococci in the gut and in some localized infections of patients, this same activity may be detrimental to enterococci in systemic infections.
Most clinical studies that investigated genotype/phenotype association of hemolytic enterococci used the presence of cylA as an indicator of hemol ysis/cytol ysis activity.While the presence of cylA gene is often associated with beta hemolysis, some cylA positiv e str ains ar e not hemol ytic and str ains that ar e cylA negative can exhibit beta hemolysis (Chai et al. 2019, Iseppi et al. 2020 ).We used CylL(L) (RS02570) and CylL(S) (RS02575) of E. faecalis V583 to determine the pr e v alence of these peptides.Both CylL(L) and CylL(S) were found in about 27% of E. f aecalis str ains but in no other enterococci (Fig. 2 ).Apart from cytolysin genes, the core genome of E. faecalis harbors three genes annotated as or with significant similarity to hemolysins, (OG1RF_RS07205, OG1RF_RS02335, and OG1RF_RS03705) but none of these gene pr oducts hav e been c har acterized.OG1RF_RS07205 encodes a putativ e hemol ysin III (Hl yIII) sharing ∼69% amino acid similarity with hemolysin III of Bacillus cereus , which was shown to oligomerize and create pores at the surface of red blood cells (Baida and Kuzmin 1996 ).OG1RF_RS02335 is annotated as hlyC , a putativ e tr ansporter involv ed in the export of hemolysins and other molecules.OG1RF_RS03705 belongs to the Tl yA famil y rRNA (cytidine-2`-O)-methyltr ansfer ase and is annotated as hemolysin A with homologues found in B. cereus , Streptococcus agalactiae , and Streptococcus sanguinis.Genes coding for Hl yIII, Hl yC, and Hl yA ar e found at high pr e v alence in all 25 species of enterococci used in our searches (75% or more strains within a given species) and can be also found in nearly all Vagococci and Tetragenococci.

Concluding remarks
The purpose of this review was to provide a comprehensive ov ervie w of the current understanding of the significance of heme in enter ococci.Equall y important w as to identify existing kno wledge gaps in the field and offer a r oadma p for futur e inv estigations.Because E. faecalis is by far the most studied species of the genus, we searched for E. faecalis hemoproteins in other enter ococci to hav e a better perspectiv e of the div ersity and importance of heme within the genus.We found that se v er al of these hemoproteins, including the w ell-kno wn catalase h and CydABCD ar e nearl y ubiquitous to E. faecalis but completely absent or part of the accessory genome of other enterococci.We suspect that the une v en distribution of known and suspected hemopr oteins among the enterococci hints at crucial differences in their evolutionary history and the different selective pressures within envir onments encounter ed by ancestr al str ains.A notable observation that arose from these comparisons was the absence of cytoc hr omes and heme-dependent catalase in E. faecium (Baureder and Hederstedt 2013 ) considering that both E. faecalis and E. f aecium r eside in the human gut and are capable of causing multiple opportunistic infections.Despite E. faecium exhibiting a higher pr e v alence for v ancomycin/m ultidrug r esistance, E. f aecalis is more abundant in the gut and far more prevalent in human infections (Arias andMurray 2012 , Huycke 2014 ).Furthermore, E. faecalis has demonstrated greater virulence in animal models compar ed to E. f aecium (Garsin et al. 2014, Fior e et al. 2019 ).Futur e studies could investigate whether these differences are, at least in part, due to E. faecalis' enhanced ability to exploit host heme to its adv anta ge.